Enantio- and Substrate-Selective Recognition of Chiral Neurotransmitters with C3-Symmetric Switchable Receptors.

We report on the synthesis of C3-symmetric enantiopure cage molecules 1, which exhibit remarkable to exclusive enantioselective recognition properties toward chiral ammonium neurotransmitters. Strong changes in the substrate selectivity are also observed when different stereoisomers of 1 are used. Furthermore, protonation/deprotonation induces a reversible modification of the conformation of 1, which switches from an imploded to an inflated form, leading to ejection and reuptake of the guest initially encaged inside the cavity.

Assessment of false transmitters as treatments for nerve agent poisoning.

Nerve agents inhibit acetylcholinesterase (AChE), leading to a build-up of acetylcholine (ACh) and overstimulation at cholinergic synapses. Current post-exposure nerve agent treatment includes atropine to treat overstimulation at muscarinic synapses, a benzodiazepine anti-convulsant, and an oxime to restore the function of AChE. Aside from the oxime, the components do not act directly to reduce the overstimulation at nicotinic synapses. The false transmitters acetylmonoethylcholine (AMECh) and acetyldiethylcholine (ADECh) are analogs of ACh, synthesised similarly at synapses. AMECh and ADECh are partial agonists, with reduced activity compared to ACh, so it was hypothesised the false transmitters could reduce overstimulation. Synthetic routes to AMECh and ADECh, and their precursors, monoethylcholine (MECh) and diethylcholine (DECh), were devised, allowing them to be produced easily on a laboratory-scale. The mechanism of action of the false transmitters was investigated in vitro. AMECh acted as a partial agonist at human muscarinic (M1 and M3) and muscle-type nicotinic receptors, and ADECh was a partial agonist only at certain muscarinic subtypes. Their precursors acted as antagonists at muscle-type nicotinic, but not muscarinic receptors. Administration of MECh and DECh improved neuromuscular function in the soman-exposed guinea-pig hemi-diaphragm preparation. False transmitters may therefore help reduce nerve agent induced overstimulation at cholinergic synapses.

Synthetic Photoswitchable Neurotransmitters Based on Bridged Azobenzenes.

Photoswitchable neurotransmitters of ionotropic kainate receptors were synthesized by tethering a glutamate moiety to disubstituted C2-bridged azobenzenes, which were prepared through a novel methodology that allows access to diazocines with higher yields and versatility. Because of the singular properties of these photochromes, photoisomerizable compounds were obtained with larger thermal stability for their inert cis isomer than for their biologically activity trans state. This enabled selective neuronal firing upon irradiation without background activity in the dark.

Recent advances of the Povarov reaction in medicinal chemistry.

The Povarov multicomponent reaction consists on the condensation of an aniline, an aldehyde, and an activated olefin to generate a tetrahydroquinoline adduct with 3 diversity points. Hereby, we report the main features of this transformation and its uses in medicinal chemistry. Relevant examples of the impact of Povarov adducts in different therapeutic areas are provided.

Cryochemical Synthesis of Polymorphous Nanostructures of a Steroid Neurohormone.

A new cryochemical strategy of producing nanoparticles and polymorphous nanostructures of drugs is used, which is based on the dynamic combination of high and low temperatures, gas and solid phases, and inert carrier gases. This technology is applied to the synthesis of nanoparticles of steroid neurohormone dehydroepiandrosterone (DHEA). We have optimized the conditions of synthesis of the new polymorphous DHEA structure, FVII. The molecules of DHEA in FVII structure are bound by hydrogen bonds via oxygen atoms. The grain size is 100 nm. It is shown that the yield and ratio of the resulting nanoforms of this hormone are determined by the nature and properties of the inert carrier gas. The highest yield and selectivity of FVII are observed when carbon dioxide is used as the carrier gas. In the case of helium, the FVII content decreases from 85 to 30% and other structures are formed. In experiments without carrier gas, nanoparticles are formed but no FVII is produced. The selectivity and the effect of carrier gas are considered on the basis of homogeneous and heterogeneous formation of nanoparticles and the relationship between particle selectivity and its activity. The synthesis of various polymorphous structures on the nanoscale is assumed to be the manifestation of the size effect in the synthesis of drugs.

Calixarene-mediated liquid membrane transport of choline conjugates 3: The effect of handle variation on neurotransmitter transport.

Upper rim phosphonic acid functionalized calix[4]arene affects selective transport of multiple molecular payloads through a liquid membrane. The secret is in the attachment of a receptor-complementary handle to the payload. We find that the trimethylammonium ethylene group present in choline is one of several general handles for the transport of drug and drug-like species. Herein we compare the effect of handle variation against the transport of serotonin and dopamine. We find that several ionizable amine termini handles are sufficient for transport and identify two ideal candidates. Their performance is significantly enhanced in HEPES buffered solutions. This inquiry completes a series of 3 studies aimed at optimization of this strategy. In completion a new approach towards synthetic receptor mediated selective small molecule transport has emerged; future work in vesicular and cellular systems will follow.

Effect of Substitution on the Aniline Moiety of the GPR88 Agonist 2-PCCA: Synthesis, Structure-Activity Relationships, and Molecular Modeling Studies.

GPR88, an orphan receptor richly expressed in the striatum, is implicated in a number of basal ganglia-associated disorders. In order to elucidate the functions of GPR88, an in vivo probe appropriate for CNS investigation is required. We previously reported that 2-PCCA was able to modulate GPR88-mediated cAMP production through a Gαi-coupled pathway. Early structure-activity relationship (SAR) studies suggested that the aniline moiety of 2-PCCA is a suitable site for diverse modifications. Aimed at elucidating structural requirements in this region, we have designed and synthesized a series of analogues bearing a variety of substituents at the phenyl ring of the aniline moiety. Several compounds (e.g., 5j, 5o) showed improved or comparable potency, but have lower lipophilicity than 2-PCCA (clogP 6.19). These compounds provide the basis for further optimization to probe GPR88 in vivo functions. Computational studies confirmed the SAR trends and supported the notion that 4'-substituents on the biphenyl ring exit through a largely hydrophobic binding site to the extracellular loop.

Identification of 2-({[1-(4-Fluorophenyl)-5-(2-methoxyphenyl)-1H-pyrazol-3-yl]carbonyl}amino)tricyclo[3.3.1.13,7]decane-2-carboxylic Acid (NTRC-844) as a Selective Antagonist for the Rat Neurotensin Receptor Type 2.

Neurotensin receptor type 2 (NTS2) compounds display analgesic activity in animal pain models. We have identified the first high-affinity NTS2-selective antagonist (8) that is active in vivo. This study also revealed that the NTS2 FLIPR assay designation for a compound, agonist, partial agonist, and so forth, did not correlate with its in vivo activity as observed in the thermal tail-flick acute model of pain. This suggests that calcium mobilization is not the signaling pathway involved in NTS2-mediated analgesia as assessed by the thermal tail-flick model. Finally, we found a significant bias between rat and human for compound 9 in the NTS2 binding assay.

Neurosteroids: Can a 2alpha,3alpha-epoxy ring make up for the 3alpha-hydroxyl group?

Seven steroid epoxides were prepared from 5α-pregn-2-en-20-one and 5α-pregn-3-en-20-one and their side-chain derivatives. All compounds were tested in vitro for binding to γ-aminobutyric acid (GABAA) receptor, some of them also in vivo for anticonvulsant action. 2α,3α-Epoxy-5α-pregnan-20-one inhibited the TBPS binding to the GABAA receptor and showed a moderate anticonvulsant action in immature rats. In contrast, its 3α,4α-isomer was inactive. More polar epoxide derivatives, modified at the side chain were less active or inactive. Noteworthy, diol 20, the product of trans-diaxial opening of the 2α,3α-epoxide 4, was not able to inhibit the TBPS binding, showing that the activity of the epoxide is due to the compound itself and not to its hydrolytic product. The 3α-hydroxyl group is known to be essential for the GABAA receptor binding. Despite the shortness of in vivo effects which are probably due to metabolic inactivation of the products prepared, our results show that the 2α,3α-epoxy ring is another structural pattern with ability to bind the GABAAR.

Caged glutamates with π-extended 1,2-dihydronaphthalene chromophore: design, synthesis, two-photon absorption property, and photochemical reactivity.

Caging and photochemical uncaging of the excitatory neurotransmitter l-glutamate (glu) offers a potentially valuable tool for understanding the mechanisms of neuronal processes. Designing water-soluble caged glutamates with the appropriate two-photon absorption property is an attractive strategy to achieve this. This paper describes the design, synthesis, and photochemical reactivity of caged glutamates with π-extended 1,2-dihydronaphthalene structures, which possess a two-photon cross-section of ∼120 GM and an excellent buffer solubility (up to 115 mM). High yields up to 99% glutamate were observed in the photolysis of two caged glutamates. Suzuki-Miyaura cross-coupling and Buchwald-Hartwig amination were used as the key reactions to synthesize the caged compounds.

Synthesis, biophysical, and pharmacological evaluation of the melanocortin agonist AST3-88: modifications of peptide backbone at Trp 7 position lead to a potent, selective, and stable ligand of the melanocortin 4 receptor (MC4R).

The melanocortin-3 (MC3R) and melanocortin-4 (MC4R) receptors are expressed in the brain and are implicated in the regulation of food intake and energy homeostasis. The endogenous agonist ligands for these receptors (α-, ß-, γ-MSH and ACTH) are linear peptides with limited receptor subtype selectivity and metabolic stability, thus minimizing their use as probes to characterize the overlapping pharmacological and physiological functions of the melanocortin receptor subtypes. In the present study, an engineered template, in which the peptide backbone was modified by a heterocyclic reverse turn mimetic at the Trp(7) residue, was synthesized using solid phase peptide synthesis and characterized by a ß-galactosidase cAMP based reporter gene assay. The functional assay identified a ∼5 nM mouse MC4R agonist (AST3-88) with more than 50-fold selectivity over the mMC3R. Biophysical studies (2D (1)H NMR spectroscopy and molecular dynamics) of AST3-88 identified a type VIII ß-turn secondary structure spanning the pharmacophore domain stabilized by the intramolecular interactions between the side chains of the His and Trp residues. Enzymatic studies of AST3-88 revealed enhanced stability of AST3-88 over the α-MSH endogenous peptide in rat serum. Upon central administration of AST3-88 into rats, a decreased food intake response was observed. This is the first study to probe the in vivo physiological activity of this engineered peptide-heterocycle template. These findings advance the present knowledge of pharmacophore design for potent, selective, and metabolically stable melanocortin ligands.

Identification of neuropeptide S antagonists: structure-activity relationship studies, X-ray crystallography, and in vivo evaluation.

Modulation of the neuropeptide S (NPS) system has been linked to a variety of CNS disorders such as panic disorder, anxiety, sleeping disorders, asthma, obesity, PTSD, and substance abuse. In this study, a series of diphenyltetrahydro-1H-oxazolo[3,4-α]pyrazin-3(5H)-ones were synthesized and evaluated for antagonist activity at the neuropeptide S receptor. The absolute configuration was determined by chiral resolution of the key synthetic intermediate, followed by analysis of one of the individual enantiomers by X-ray crystallography. The R isomer was then converted to a biologically active compound (34) that had a Ke of 36 nM. The most potent compound displayed enhanced aqueous solubility compared with the prototypical antagonist SHA-68 and demonstrated favorable pharmacokinetic properties for behavioral assessment. In vivo analysis in mice indicated a significant blockade of NPS induced locomotor activity at an ip dose of 50 mg/kg. This suggests that analogs having improved drug-like properties will facilitate more detailed studies of the neuropeptide S receptor system.

Synthesis and biological evaluation of bis-CNB-GABA, a photoactivatable neurotransmitter with low receptor interference and chemical two-photon uncaging properties.

Photoactivatable "caged" neurotransmitters allow optical control of neural tissue with high spatial and temporal precision. However, the development of caged versions of the chief vertebrate inhibitory neurotransmitter, γ-amino butyric acid (GABA), has been limited by the propensity of caged GABAs to interact with GABA receptors. We describe herein the synthesis and application of a practically useful doubly caged GABA analog, termed bis-α-carboxy-2-nitrobenzyl-GABA (bis-CNB-GABA). Uncaging of bis-CNB-GABA evokes inward GABAergic currents in cerebellar molecular layer interneurons with rise times of 2 ms, comparable to flash duration. Response amplitudes depend on the square of flash intensity, as expected for a chemical two-photon uncaging effect. Importantly, prior to uncaging, bis-CNB-GABA is inactive at the GABAA receptor, evoking no changes in holding current in voltage-clamped neurons and showing an IC50 of at least 2.5 mM as measured using spontaneous GABAergic synaptic currents. Bis-CNB-GABA is stable in solution, with an estimated half-life of 98 days in the light. We expect that bis-CNB-GABA will prove to be an effective tool for high-resolution chemical control of brain circuits.

The development of benzo- and naphtho-fused quinoline-2,4-dicarboxylic acids as vesicular glutamate transporter (VGLUT) inhibitors reveals a possible role for neuroactive steroids.

Substituted quinoline-2,4-dicarboxylates (QDCs) are conformationally-restricted mimics of glutamate that were previously reported to selectively block the glutamate vesicular transporters (VGLUTs). We find that expanding the QDC scaffold to benzoquinoline dicarboxylic acids (BQDC) and naphthoquinoline dicarboxylic acids (NQDCs) improves inhibitory activity with the NQDCs showing IC50∼70 µM. Modeling overlay studies showed that the polycyclic QDCs resembled steroid structures and led to the identification and testing of estrone sulfate, pregnenolone sulfate and pregnanolone sulfate that blocked the uptake of l-Glu by 50%, 70% and 85% of control, respectively. Pregnanolone sulfate was further characterized by kinetic pharmacological determinations that demonstrated competitive inhibition and a Ki of ≈20 µM.

Neurosteroid analogues. 18. Structure-activity studies of ent-steroid potentiators of γ-aminobutyric acid type A receptors and comparison of their activities with those of alphaxalone and allopregnanolone.

A model of the alignment of neurosteroids and ent-neurosteroids at the same binding site on γ-aminobutyric acid type A (GABAA) receptors was evaluated for its ability to identify the structural features in ent-neurosteroids that enhance their activity as positive allosteric modulators of this receptor. Structural features that were identified included: (1) a ketone group at position C-16, (2) an axial 4α-OMe group, and (3) a C-18 methyl group. Two ent-steroids were identified that were more potent than the anesthetic steroid alphaxalone in their threshold for and duration of loss of the righting reflex in mice. In tadpoles, loss of righting reflex for these two ent-steroids occurs with EC50 values similar to those found for allopregnanolone. The results indicate that ent-steroids have considerable potential to be developed as anesthetic agents and as drugs to treat brain disorders that are ameliorated by positive allosteric modulators of GABAA receptor function.

Aminoindanes--the next wave of 'legal highs'?

Due to its closed ring system, 2-aminoindane is a conformationally rigid analogue of amphetamine. Internet websites offering synthetic compounds as 'research chemicals' have recently been advertising 5,6-methylenedioxy-2-aminoindane (MDAI), 5, 6-methylenedioxy-N-methyl-2-aminoindane (MDMAI), 5-iodo-2-aminoindane (5-IAI), and 5-methoxy-6-methyl-2-aminoindane (MMAI). The chemistry, pharmacology, and toxicological aspects of this new class of psychoactive substances are reviewed, as these could become the next wave of 'legal highs'.

Generation of novel radiolabeled opiates through site-selective iodination.

Tritiated opioid radioligands have proven valuable in exploring opioid binding sites. However, tritium has many limitations. Its low specific activity and limited counting efficiency makes it difficult to examine low abundant, high affinity sites and its disposal is problematic due to the need to use organic scintillants and its relatively long half-life. To overcome these issues, we have synthesized both unlabeled and carrier-free radioiodinated iodobenzoyl derivatives of 6ß-naltrexamine ((125)I-BNtxA, 18), 6ß-naloxamine ((125)I-BNalA, 19) and 6ß-oxymorphamine ((125)I-BOxyA, 20) with specific activities of 2100Ci/mmol. To optimize the utility of the radioligand, we designed a synthesis in which the radiolabel is incorporated in the last synthetic step, which required the selective iodination of the benzoyl moiety without incorporation into the phenolic A ring. Competition studies demonstrated high affinity of the unlabelled compounds for opioid receptors in transfected cell lines, as did the direct binding of the (125)I-ligands to the opioid receptors. The radioligand displayed very high sensitivity, enabling a marked reduction in tissue, as well as excellent signal/noise characteristics. These new (125)I-radioligands should prove valuable in future studies of opioid binding sites.

The use of symmetry in enantioselective synthesis: four pairs of chrysene enantiomers prepared from 19-nortestosterone.

Expansion of the D-ring of 19-norsteroids with incorporation of the steroid C-18 methyl group into a newly formed six-membered ring provides easy access to the chrysene ring system. By taking advantage of the symmetry of the chrysene ring system and avoiding meso chrysene intermediates, four optically pure 2,8-difunctionalized (C-2 hydroxyl group and C-8 oxo group) hexadecahydrochrysene diastereomers, and their corresponding optically pure enantiomers were prepared from 19-nortestosterone. The eight chrysene stereoisomers are of interest as starting materials for preparing chrysene analogues of physiologically important neurosteroids.